Nucleic acid based microbial diagnostic techniques - QBRDA

1. N U C L E I C A C I D B A S E D M I C R O B I A L
D I A G N O S T I C T E C H N I Q U E S

2.  Nucleic acid-based diagnostics detect the presence of a pathogen either by
directly detecting the presence of DNA or RNA nucleic acids in the host or by
first amplifying the pathogen DNA or RNA.
Nucleic acid-based diagnostics are a standard central laboratory technique,
although simplified nucleic acid-based diagnostics that may be useful in
resource-poor settings are emerging.
Nucleic acid-based diagnostics detect specific nucleic acid (i.e. DNA or RNA).
In the case of infectious diseases, nucleic acid-based diagnostics detect DNA or
RNA from the infecting organism.
For non-infectious diseases, nucleic acid-based diagnostics may be used to
detect a specific gene or the expression of a gene associated with disease.

3. COMMON NUCLEIC-ACID BASED DIAGNOSTIC
TECHNIQUES USED TO DIAGNOSE INFECTIOUS DISEASES
Description Strengths Weaknesses
PCR/RT-PCR DNA (or RNA reverse
transcribed to cDNA) is
amplified to detect the
presence of a pathogen or
host gene of interest
DNA is amplified which
allows for sensitive
detection
Requires highly skilled
technician and laboratory
equipment, amplified
material can contaminate
subsequent samples
Isothermal amplification Similar to PCR, but uses
simplified amplification
techniques that operate at a
single temperature
DNA is amplified which
allows for sensitive
detection, simplified
isothermal amplification is
more amenable to point-of-
care devices/low-resource
settings
Newer technology that has
not been extensively
clinically validated,
amplified material can
contaminate subsequent
samples

4. Hybridization DNA probes bind directly to
DNA of pathogen or host
gene of interest
No risk of contamination of
new samples with amplified
material
Because the target DNA is
not amplified, technique can
be less sensitive, requires
highly skilled technician and
laboratory equipment
Sequencing The specific genetic code is
determined for an amplified
piece of DNA or the full
genome of an organism
Beyond confirming the
presence of a pathogen,
sequencing can provide
detailed information about
the original and unique
characteristics of the
pathogen in a specific
patient, not practical for
low-resource settings
Sequencing techniques are
expensive, time consuming,
and require both a highly
skilled technician and
expensive laboratory
equipment

5. QBRDA
QBRDA- Q-beta replication dependant amplification
A sensitive, non isotopic hybridization assay termed "dual capture" is described. The assay
rapidly and specifically detects very low levels of target nucleic acids and organisms.
The assay is based on the principles of sandwich hybridization, reversible target capture, and Q-
Beta replicase amplification.
The assay can be completed in less than 4 h, and in the described model format, it detects
Chlamydia trachomatis rRNA or rDNA. Up to 96 samples can be analyzed simultaneously.
The assay employs two types of probes: a test-specific capture probe, which mediates the
cycling of the target probe complex on and off derivatized magnetic beads, and a replicatable
RNA detector molecule containing a sequence complementary to and adjacent to the capture
probe site on the target.

6. Following reversible target capture, detection of the signal is accomplished by
replication of the detector molecule by Q-Beta replicase in the presence of
propidium iodide.
A specific assay signal can be detected from as few as 1,000 molecules above
the background.
In a limited study of 94 urogenital samples the assay detected five of the six
culture-positive samples and did not detect the C. trachomatis target in 85 of
the 88 culture-negative samples.